Fluorescent group-containing carbodiimide compound precursor, fluorescent group-containing carbodiimide compound and methods for producing them

ABSTRACT

A novel fluorescent group-containing carbodiimide compound having at least one group selected from a carboxyl group, a sulfo group, a phosphono group and a phospho group which have substitution of an alkali metal, an alkaline earth metal or a basic group containing a nitrogen or phosphorus atom, which is prepared by using a novel fluorescent group-containing carbodiimide compound precursor having a halogen atom or a sulfonic acid group; and a method for detecting a nucleic acid by hybridization utilizing a nucleic acid labeled with a labeling substance, which utilizes the fluorescent group-containing carbodiimide compound as the labeling substance.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/533,918, filed Mar. 23, 2000 now U.S. Pat. No. 6,642,380.

BACKGOUND OF THE INVENTION

The present invention relates to a novel fluorescent group-containingcarbodiimide compound precursor and a fluorescent group-containingcarbodiimide compound. More specifically, it relates to a fluorescentgroup-containing carbodiimide compound precursor that contains a halogenatom or a sulfonic acid group, which enables easy and efficient bondingof a fluorescent group and a method for producing it, as well as afluorescent group-containing carbodiimide compound which has at leastone group selected from a carboxyl group, a sulfo group, a phosphonogroup and a phospho group, which enables easy and efficient introductionof a fluorescent group into nucleic acids and proteins, a method forproducing it and an analysis method utilizing it.

As methods for introducing a fluorescent substance into nucleic acids,there have been known, for example, introduction of a fluorescentsubstance into a nucleic acid by using a nucleotide bonded to afluorescent substance through an enzymatic reaction and so forth(Japanese Patent Application Application Laid-open No. 6-271599),bonding of streptavidin, to which a fluorescent substance has alreadybeen bonded, to a biotin-labeled nucleic acid, a reaction of afluorescent substance containing an amine-reactive group with a nucleicacid bonded to an amino-linker and so forth. In addition, there are alsomethods other than the above, such as utilization of a fluorescentgroup-containing carbodiimide compound which is highly reactive tonucleic acid bases (Japanese Patent Application Application Laid-openNo. 10-287870).

However, among the aforementioned methods, the method described inJapanese Patent Application Laid-open No. 6-271599, the method utilizingbiotin labeling and the method utilizing amino-linker bonding havedrawbacks that it is not possible to introduce the fluorescent substanceinto naturally occurring nucleic acids, and the operations arecomplicated. The fluorescent group-containing carbodiimide compoundexhibit good solubility in water because of the existence of quaternaryammonium salt in its molecular structure. However, in order to utilize afluorescent substance with low solubility in water, such as afluorescent substance having such a large electron system that it shouldhave absorption in the near-infrared region, further improvement of thewater solubility has been desired.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a novel fluorescentgroup-containing carbodiimide compound, which enables efficient and easyintroduction of fluorescent groups into proteins and nucleic acidsincluding naturally occurring nucleic acids, and exhibits sufficientwater solubility, and to provide a fluorescent group-containingcarbodiimide compound precursor, which can be easily and efficientlybonded to a fluorescent group, and is suitable for the production of theaforementioned fluorescent group-containing carbodiimide compound.

The inventors of the present invention earnestly studied in order toachieve the aforementioned object. As a result, they found that afluorescent group-containing carbodiimide compound having at least onegroup selected form a carboxyl group, a sulfo group, a phosphono groupand a phospho group with sufficient water solubility could be producedby starting from a carbodiimide compound having a halogen atom or asulfonic acid group, and that a fluorescent group could be easily andefficiently introduced into nucleic acids including naturally occurringnucleic acids and proteins by utilizing the aforementioned fluorescentgroup-containing carbodiimide compound. Thus, they accomplished thepresent invention.

The present invention provides the followings.

-   (1) A fluorescent group-containing carbodiimide compound precursor    having a halogen atom or a sulfonic acid group which is represented    by the following general formula (I):    B—Y³—N═C═N—Y²—W—Y¹-[A_(n)]-Y⁴—X  (I)    wherein,

X represents a halogen atom or a sulfonic acid group;

A represents a functional group which is selected from the groupconsisting of —CH₂—, —NHCO—, —CONH—, —O—, —S—, —NR¹— wherein R¹represents a linear, cyclic or branched saturated or unsaturatedaliphatic hydrocarbon group having 1–20 carbon atoms, —NR²R³— wherein R²and R³ independently represent a hydrogen atom, a linear or branchedsaturated or unsaturated aliphatic hydrocarbon group having 1–20 carbonatoms, or a cycloalkyl group, an aryl group or an aralkyl group whichmay have a substituent, provided that when one of R² and R³ is ahydrogen atom, the other represents a linear or branched saturated orunsaturated aliphatic hydrocarbon group having 1–20 carbon atoms, or acycloalkyl group, an aryl group or an aralkyl group which may have asubstituent, or R² and R³ may be bonded to each other to form as a wholea nitrogen-containing heterocyclic group which may contain an oxygenatom, —COO—, —OCO—, —NHSO₂—, —NHC(S)NH— and —SO₂NH—;

n represents 0 or 1;

W represents a direct bond or a quaternary onium group;

Y¹, Y², Y³ and Y⁴ each independently represent a functional grouprepresented by the general formula (II):—(CH₃)_(p)-(L)_(r)-(CH₂)_(q)—  (II)wherein, L represents a functional group selected from the groupconsisting of —CH₂—, —NHCO—, —CONH—, —O—, —S—, —NR¹— wherein R¹ has thesame meaning as defined for the formula (I), —NR²R³— wherein R² and R³have the same meanings as defined for the formula (I), —COO—, —OCO—,—NHSO₂—, —NHC(S)NH— and —SO₂NH—; p and q each independently represent aninteger of from 0 to 20, and r represents 0 or 1;

B represents a hydrogen atom or a monovalent organic group being eitherthe same as or different from —W—Y¹-[A]_(n)-Y⁴—X in the formula (I); and

any of the functional groups represented by B, Y¹, Y², Y³, Y⁴, A or Wmay have a group selected from a carboxyl group, a sulfo group, aphosphono group or a phospho group which have substitution of an alkalimetal, an alkaline earth metal or a basic group containing a nitrogen orphosphorus atom.

-   (2) A fluorescent group-containing carbodiimide compound having at    least one group selected from a carboxyl group, a sulfo group, a    phosphono group and a phospho group which have substitution of an    alkali metal, an alkaline earth metal or a basic group containing a    nitrogen or phosphorus atom, which is represented by the following    general formula (III):

wherein,

B, Y¹, Y², Y³, Y⁴, A, X, W and n have the same meanings as defined forthe general formula (I);

F represents a fluorescent group;

Q represents either a tertiary or quaternary nitrogen atom, or atertiary or quaternary phosphorus atom;

R⁴ and R⁵ each independently represent a hydrogen atom, a linear orbranched saturated or unsaturated aliphatic hydrocarbon group having1–20 carbon atoms, or a cycloalkyl group, an aryl group or an aralkylgroup which may contain a substituent, provided that when one of R⁴ andR⁵ is a hydrogen atom, the other represents a linear or branchedsaturated or unsaturated aliphatic hydrocarbon group having 1–20 carbonatoms, or a cycloalkyl group, an aryl group or an aralkyl group whichmay contain a substituent, or R⁴ and R⁵ may be bonded to each other toform a nitrogen-containing heterocyclic group or a phosphorus-containingheterocyclic group, which may contain an oxygen atom, as -Q⁺R⁴R⁵—;

Y⁵ has the same meaning as defined for Y¹, Y², Y³ and Y⁴;

at least one functional group selected from B, Y¹, Y², Y³, Y⁴, Y⁵, A, W,R⁴, R⁵ and F has at least one group selected from a carboxyl group, asulfo group, a phosphono group and a phospho group which havesubstitution of an alkali metal, an alkaline earth metal or a basicgroup containing a nitrogen or phosphorus atom.

-   (3) The fluorescent group-containing carbodiimide compound precursor    according to (1), wherein at least one functional group selected    from B, Y¹, Y², Y³, Y⁴, A and W in the formula (I) has at least one    group selected from a carboxyl group, a sulfo group, a phosphono    group and a phospho group which have substitution of an alkali    metal, an alkaline earth metal or a basic group containing a    nitrogen or phosphorus atom.-   (4) The fluorescent group-containing carbodiimide compound according    to (2), wherein at least one functional group selected from B, Y¹,    Y², Y³, Y⁴, A and W in the formula (III) has at least one group    selected from a carboxyl group, a sulfo group, a phosphono group and    a phospho group which have substitution of an alkali metal, an    alkaline earth metal or a basic group containing a nitrogen or    phosphorus atom.-   (5) The fluorescent group-containing carbodiimide compound according    to (2), wherein at least one functional group selected from Y⁵, R⁴,    R⁵ and F in the formula (III) has at least one group selected from a    carboxyl group, a sulfo group, a phosphono group and a phospho group    which have substitution of an alkali metal, an alkaline earth metal    or a basic group containing a nitrogen or phosphorus atom.-   (6) A method for producing the fluorescent group-containing    carbodiimide compound precursor as defined in (1), which comprises    the following steps (A), (B) and (C):-   (A) a step of synthesizing a (thio)urea compound represented by the    following general formula (VI) through a reaction of an    iso(thio)cyanate compound represented by the following general    formula (IV) with an amine compound represented by the following    general formula (V):

wherein, B, Y¹, Y², Y³, Y⁴, A, W and n have the same meanings as definedfor the aforementioned formula (I); T represents an oxygen atom or asulfur atom; and Z represents a hydroxyl group, an alkyl group, analkenyl group, a vinyl group, an allyl group, a phenyl group, a carboxylgroup, a sulfonyl group-containing derivative group or a phosphoniumgroup;

-   (B) a step of halogenating or sulfonating the compound represented    by the general formula (VI) obtained in the aforementioned step (A)    to prepare a compound represented by the following general formula    (VII):

wherein, B, Y¹, Y², Y³, Y⁴, W, A, n and X have the same meanings asdefined for the formula (I); and T represents an oxygen atom and or asulfur atom;

-   (C) a step of carbodiimidating the compound represented by the    following general formula (VII) obtained in the aforementioned    step (B) by dehydration or oxidative desulfurization reaction.-   (7) The production method according to (6), which produces the    fluorescent group-containing carbodiimide compound precursor as    defined in (3), wherein at least one functional group selected from    B, Y¹, Y², Y³, Y⁴, A and W has at least one group selected from a    carboxyl group, a sulfo group, a phosphono group and a phospho group    which have substitution of an alkali metal, an alkaline earth metal    or a basic group containing a nitrogen or phosphorus atom.-   (8) A method for producing the fluorescent group-containing    carbodiimide compound as defined in (2), which comprises a step of    selecting a carbodiimide compound and a fluoresent group-containing    compound from the fluorescent group-containing carbodiimide compound    precursor as defined in (1) and a fluorescent group-containing    compound represented by the following general formula (VIII):    Q′R⁴R⁵—Y⁵⁻F  (VIII)    wherein, R⁴, R⁵, Y⁵ and F have the same meanings as defined for the    general formula (III), and Q′ represents a secondary or tertiary    nitrogen-containing group or a secondary or tertiary    phosphorus-containing group which may have substitution of a group    selected from a hydroxyl group, an alkyl group, an alkenyl group, a    vinyl group, an allyl group, a phenyl group, a carboxyl group, a    sulfonyl group-containing derivative group and a phosphonium group,    respectively, so that at least one of the carbodidimide compound and    the fluorescent group-containing compound should have a functional    group selected from a carboxyl group, a sulfo group, a phosphono    group and a phospho group which have substitution of an alkali    metal, an alkaline earth metal or a basic group containing a    nitrogen or phosphorus atom, to allow the carbodidimide compound and    the fluorescent group-containing compound to react with each other.-   (9) The production method according to (8) for producing the    fluorescent group-containing carbodiimide compound according to (4),    wherein at least one functional group selected from B, Y¹, Y², Y³,    Y⁴, A and W has at least one group selected from a carboxyl group, a    sulfo group, a phosphono group or a phospho group which are    substituted with an alkali metal, an alkaline earth metal or a basic    group containing a nitrogen or phosphorus atom.-   (10) The production method according to (8), which produces the    fluorescent group-containing carbodiimide compound as defined in    (5), wherein at least one functional group selected from Y⁵, R⁴, R⁵    and F has at least one group selected from a carboxyl group, a sulfo    group, a phosphono group or a phospho group which are substituted    with an alkali metal, an alkaline earth metal or a basic group    containing a nitrogen or phosphorus atom.-   (11) A method for detecting a nucleic acid by hybridization    utilizing a nucleic acid labeled with a labeling substance, wherein    the fluorescent group-containing carbodiimide compound as defined    in (2) is used as the labeling substance.-   (12) The method according to (11), wherein the fluorescent    group-containing carbodiimide compound as defined in (4) as is used    the labeling substance.-   (13) The method according to (11), wherein the fluorescent    group-containing carbodiimide compound as defined in (5) as is used    the labeling substance.

The fluorescent group-containing carbodiimide compound of the presentinvention exhibits sufficient water solubility, and use of it enablesefficient and easy introduction of fluorescent groups into proteins andnucleic acids. Furthermore, the fluorescent group-containingcarbodiimide compound precursor of the present invention is particularlysuitable for the production of the aforementioned fluorescentgroup-containing carbodiimide compound, since it can be easily andefficiently bonded to a fluorescent group.

The term “iso(thio)cyanate compound” used herein refers to an isocyanatecompound or an isothiocyanate compound. The term “(thio)urea compound”used herein refers to a urea compound or a thiourea compound. The term“sulfonyl group-containing derivative group” used herein refers to amonovalent group created when one of the valences of a divalent sulfonylmolecule is used to bond with an atom or a group. Examples of thesulfonyl group-containing derivative group are alkylsulfonyl groups andalkoxyphenyl groups.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will be described hereafter. Thefluorescent group-containing carbodiimide compound of the presentinvention will be explained first.

(1) Fluorescent Group-Containing Carbodiimide Compound

The fluorescent group-containing carbodiimide compound of the presentinvention having at least one group selected from a carboxyl group, asulfo group, a phosphono group and a phospho group which havesubstitution of an alkali metal, an alkaline earth metal or a basicgroup containing a nitrogen or phosphorus atom is a compound representedby the general formula (III). The method for producing this compound isnot particularly limited. For example, it can be produced by selecting acarbodiimide compound and a fluorescent group-containing compound from afluorescent group-containing carbodidimide compound precursor having ahalogen atom and a sulfonic acid group represented by the generalformula (I) and a fluorescent group-containing compound represented bythe general formula (VIII), respectively, so that at least one of thecarbodidimide compound and the fluorescent group-containing compoundshould have a functional group selected from a carboxyl group, a sulfogroup, a phosphono group and a phospho group which have substitution ofan alkali metal, an alkaline earth metal or a basic group containing anitrogen or phosphorus atom, to allow them to reaction with each other.

(i) Fluorescent Group-Containing Carbodiimide Compound Precursor

According to the present invention, the fluorescent group-containingcarbodiimide compound precursor having a halogen atom or a sulfonic acidgroup has a structure represented by the general formula (I).

X in the formula (I) is not particularly limited so long as it is eithera halogen atom or a sulfonic acid group, and known ones can be used. Inparticular, Cl, Br or I atoms are preferable as the halogen atom becauseof reactivity. As the sulfonic acid group, a tosyl group and a mesylgroup are preferable because of reactivity.

In the formula (I), A represents a functional group selected from thegroup consisting of —CH₂—, —NHCO—, —CONH—, —O—, —S—, —NR¹—, —NR²R³—,—COO—, —OCO—, —NHSO₂—, —NHC(S)NH— and —SO₂NH—, and n represents 0 or 1.

R¹ represents a linear, cyclic or branched saturated or unsaturatedaliphatic hydrocarbon group having 1–20 carbon atoms. R² and R³ eachindependently represent a hydrogen atom, a linear or branched saturatedor unsaturated aliphatic hydrocarbon groups having 1–20 carbon atoms, ora cycloalkyl group, an aryl group or an aralkyl group which may have asubstituent. However, when one of R² and R³ is a hydrogen atom, theother represents a linear or branched saturated or unsaturated aliphatichydrocarbon group having 1–20 carbon atoms, or a cycloalkyl group, anaryl group, or an aralkyl group which may have a substituent. Further,R² and R³ may be bonded to each other to form as a whole anitrogen-containing heterocyclic group which may contain an oxygen atom.As described above, n represents 0 or 1, and thus the compoundrepresented by the general formula (I) may or may not have A.

A group selected from a carboxyl group, a sulfo group, a phosphono groupand a phospho group which have substitution of an alkali metal, analkaline earth metal or a basic group containing a nitrogen orphosphorus atom may be substituted for any replaceable hydrogen atom ofA.

W represents a direct bond or a quaternary onium group. The term“quaternary onium group” used herein refers to a group which can form aquaternary onium salt when it is introduced into the compound of theformula (I). Specific examples of such a quaternary onium group includethose groups represented by the following general formulae (IX), (X) and(XI):

wherein, R⁶, R⁷, R⁸ and R⁹ each independently represent a linear orbranched saturated or unsaturated aliphatic hydrocarbon group having1–20 carbon atoms, a cycloalkyl group, an aryl group or aralkyl grouphaving 6–20 carbon atoms. These hydrocarbon groups may have substitutionof a carboxyl group, a sulfonyl group-containing derivative group, aphosphonium group, a phenyl group, a cycloalkyl group, or a hydroxylgroup.

Of the above quaternary onium groups, particularly preferred groups usedin the present invention are the quaternary onium group represented bythe formula (IX) in which R⁶ and R⁷ are alkyl groups, and the grouprepresented by the general formula (X).

Furthermore, a group selected from a carboxyl group, a sulfo group, aphosphono group and a phospho group which have substitution of an alkalimetal, an alkaline earth metal or a basic group containing a nitrogen orphosphorus atom may be substituted for any replaceable hydrogen atom inW.

Y¹, Y², Y³ and Y⁴ are linkers which link carbodiimide groups to B, A orX, and each independently represent a functional group represented bythe formula (II). In the general formula (II), L represents a functionalgroup selected from the group consisting of —CH₂—, —NHCO—, —CONH—, —O—,—S—, —NR¹—, —NR²R³—, —COO—, —OCO—, —NHSO₂—, —NHC(S)NH— and —SO₂NH—; pand q each independently represent an integer of from 0 to 20; and rrepresents 0 or 1. R¹, R² and R³ have the same meanings as defined forthe formula (I).

A direct bond or an alkylene group that has 1–10 carbon atoms in itsmain chain and may have a methyl group as a side chain may preferably beused as Y¹, Y² Y³ and Y⁴. This alkylene group is not particularlylimited, and known ones can be used. Y¹, Y², Y³ and Y⁴ may be thealkylene groups bonded through a functional group selected from thegroup consisting of —NHCO—, —CONH—, —O—, —S—, —NR¹—, —NR²R³—, —COO—,—OCO—, —NHSO₂—, —NHC(S)NH— and —SO₂NH—.

Furthermore, a group selected form a carboxyl group, a sulfo group, aphosphono group or a phospho group which have substitution of an alkalimetal, an alkaline earth metal or a basic group containing a nitrogen orphosphorus atom may be substituted for any replaceable hydrogen atompresent in each of Y¹, Y², Y³ and Y⁴.

B represents a hydrogen atom or a monovalent organic group. Thismonovalent organic group may be either the same as, or different from—W—Y¹-[A]_(n)-Y⁴—X in the formula (I). It is preferably an alkyl group,a tertiary amino group or a quaternary ammonium group, and preferredexamples thereof are listed below.

-   (a) A nitrogen-containing heterocyclic group in which a nitrogen    atom may be quaternarized with a hydrogen atom, a saturated or    unsaturated aliphatic hydrocarbon group, an aryl group, an aralkyl    group, or a fluorescent group-containing organic group, such as a    pyridyl group, a pyrrolidinium group, and a piperidinium group.-   (b) An amino group in which a nitrogen atom is quaternarized with a    hydrogen atom, a saturated or unsaturated aliphatic hydrocarbon    group, an aryl group, an aralkyl group, or a fluorescent    group-containing organic group, such as dimethylamino group,    diethylamino group, and diisopropylamino group.-   (c) A heterocyclic tertiary amino group or quaternary ammonium group    represented by the following general formula (XII) or (XIII):

wherein, R¹¹ and R¹² each independently represent a hydrogen atom, alinear or branched saturated or unsaturated aliphatic hydrocarbon grouphaving 1–10 carbon atoms, an aryl group or an aralkyl group; Jrepresents an anion such as a sulfate ion, an alkylsulfate ion, anarylsulfate ion, a halosulfate ion and a halide ion; R¹³ represents anoxygen atom or an ionized atom thereof, or a methylene group; and mrepresents 0 or 1.

More specifically, those groups represented by the following formulaecan be mentioned:

wherein, R¹⁴ and R¹⁵ each independently represent a hydrogen atom, analkyl group having 1–10 carbon atoms, or a phenyl group which may havesubstitution of an alkyl group having 1–10 carbon atoms.

-   (d) An alkyl group which may have a substituent, for example, a    methyl group, an ethyl group, a n-propyl group, an isopropyl group,    a n-butyl group, a sec-butyl group, a tert-butyl group and a    cyclohexyl group, an aryl group such as a phenyl group and so forth.

A group selected from a carboxyl group, a sulfo group, a phosphono groupand a phospho group which have substitution of an alkali metal, analkaline earth metal or a basic group containing a nitrogen orphosphorus atom may be substituted for any replaceable hydrogen atom ofB.

As described above, in the fluorescent group-containing carbodiimidecompound precursor of the present invention having a halogen atom or asulfonic acid group, which is represented by the general formula (I),any of the functional groups represented by B, Y¹, Y², Y³, Y⁴, A and Wmay have a group selected from a carboxyl group, a sulfo group, aphosphono group and a phospho group which have substitution of an alkalimetal, an alkaline earth metal or a basic group containing a nitrogen orphosphorus atom. However, it is preferable that at least one functionalgroup selected from these groups has at least one group selected From acarboxyl group, a sulfo group, a phosphono group and a phospho groupwhich have substitution of an alkali metal, an alkaline earth metal or abasic group containing a nitrogen or phosphorus atom.

Specific examples of the carboxyl group, the sulfo group, the phosphonogroup and the phospho group which have substitution of the alkali metal,the alkaline earth metal or the basic group containing the nitrogen orphosphorus atom are —COONa, —COOK, (—COO)₂Mg, (—COO)₂Ca, (—COO)₂Ba,—COONH₄, —COONR¹H₃, —COONR¹ ₂H₂, —COONR¹ ₃H, —COONR¹ ₄, —SO₃Na, —SO₃NH₄,—SO₃NR¹H₃, —SO₃NR¹ ₂H₂, —SO₃NR¹ ₃H, —SO₃NR¹ ₄, —PO₃Na₂, —PO₃K₂, —PO₃Ca,—PO₃(NH₄)₂, —PO₃(NR¹H₃)₂, —PO₃(NR¹ ₂H₂)₂, —PO₃(NR¹ ₃H)₂, —PO₃(NR¹ ₄)₂,—OPO₃Na₂, —OPO₃K₂, —OPO₃(NR¹ ₄)₂ and so forth, where R¹ has the samemeaning as R¹ in the formula (I).

Specific examples of the fluorescent group-containing carbodiimidecompound precursor of the present invention represented by the formula(I) include, for example,N-methyl-(3-morpholinopropyl)-N-(hexane-6-iodide)-carbodiimide,N-methyl-(3-morpholinopropyl)-N-(hexane-6-bromide)-carbodiimide,N-methyl-(3-morpholinopropyl)-N-(hexane-6-chloride)-carbodiimide,N-methyl-(3-morpholinopropyl)-N-(hexane-6-tosylate)-carbodiimide,N-methyl-(3-morpholinopropyl)-N-(hexane-6-mesylate)-carbodiimide,N-methyl-(3-morpholinopropyl)-N-propyl(sodium2-ethylcarboxylate)-3-iodide)-carbodiimide,N-methyl-(3-morpholinopropyl)-N-(propyl-(potassium2-ethylcarboxylate)-3-iodide)-carbodiimide,N-methyl-(3-morpholinopropyl)-N-(propyl-(sodium2-propyl-carboxylate)-3-iodide)-carbodiimide,N-methyl-(3-morpholinopropyl)-N-(propyl-(sodium2-isopropyl-carboxylate)-3-iodide)-carbodiimide and so forth.

(ii) Fluorescent Group-Containing Carbodiimide Compound

The fluorescent group-containing carbodiimide compound of the presentinvention, which has at least one group selected from a carboxyl group,a sulfo group, a phosphono-group and a phospho group which havesubstitution of an alkali metal, an alkaline earth metal or a basicgroup containing a nitrogen or phosphorus atom, can be obtained by, forexample, selecting a carbodiimide compound and a fluorescentgroup-containing cmpound from the fluorescent group-containingcarbodiimide compound precursor having the halogen atom or the sulfonicacid group represented by the general formula (I), and the fluorescentgroup-containing compound represented by the general formula (VIII),respectively, so that at least one of the carbodiimide compound and thefluorescent group-containing compound should have a functional groupselected from a carboxyl group, a sulfo group, a phosphono group and aphospho group which have substitution of an alkali metal, an alkalineearth metal or a basic group containing a nitrogen or phosphorus atom,to allow them to react with each other. The structure of the fluorescentgroup-containing carbodiimide compound of the present invention isrepresented by the general formula (III).

The fluorescent group F in the formula (III) is not particularlylimited, and any of known fluorescent groups can be used. Specificexamples thereof include, for example, those groups derived fromanthracene derivatives, coumarin derivatives, pyrene derivatives,perylene derivatives, dansyl derivatives, rhodamine derivatives, oxazolederivatives, benzothiazole derivatives, benzoxadiazole derivatives,boron dipyrromethene difluoride derivatives, thiazole orangederivatives, fluorescent rare earth metal compound derivatives, cyaninederivatives and so forth.

A group selected from a carboxyl group, a sulfo group, a phosphono groupand a phospho group which have substitution of an alkali metal, analkaline earth metal or a basic group containing a nitrogen orphosphorus atom may be substituted for any replaceable hydrogen atom ofthe fluorescent group.

In the formula (III), Q represents a tertiary or quaternary nitrogenatom or a tertiary or quaternary phosphorus atom. In the formula (III),R⁴ and R⁵ each independently represent a hydrogen atom, a linear orbranched saturated or unsaturated aliphatic hydrocarbon group having1–20 carbon atoms, or a cycloalkyl group, an aryl group or an aralkylgroup which may have a substituent. However, when one of R⁴ and R⁵ ishydrogen atom, the other represents a linear or branched saturated orunsaturated aliphatic hydrocarbon group having 1–20 carbon atoms, or acycloalkyl group, an aryl group or an aralkyl group which may have asubstituent. Further, R⁴ and R⁵ may be bonded to each other to form anitrogen-containing heterocyclic group or a phosphorus-containingheterocyclic group, which may contain oxygen atom, as -Q⁺ R⁴R⁵—. Thatis, the Q forms a tertiary or quaternary onium group in combination withR⁴ and R⁵ in the form of -Q⁺R⁴R⁵—. Preferred examples of the tertiary orquaternary onium group include those quaternary onium groups mentionedas preferred groups for W.

A group selected from a carboxyl group, a sulfo group, a phosphono groupand a phospho group which are substituted with an alkali metal, analkaline earth metal or a basic group containing a nitrogen orphosphorus atom may be substituted for any replaceable hydrogen atom ofR⁴ and R⁵.

In the formula (III), B, Y¹, Y², Y³, Y⁴, A, X, W, R², R³ and n have thesame meanings as defined for the general formula (I). Further, Y⁵ in theformula (III) has the same meaning as defined for Y¹, Y², Y³ and Y⁴ inthe formula (I).

As described above, in the fluorescent group-containing carbodiimidecompound represented by the general formula (III) of the presentinvention, any of the functional groups represented by B, Y¹, Y², Y³,Y⁴, Y⁵ A, W, R⁴, R⁵ and F may have a group selected from a carboxylgroup, a sulfo group, a phosphono group and a phospho group which havesubstitution of an alkali metal, an alkaline earth metal or a basicgroup containing a nitrogen or phosphorus atom. At the same time, atleast one functional group selected from these groups necessarily has atleast one group selected from a carboxyl group, a sulfo group, aphosphono group and a phospho group which are substituted with an alkalimetal, an alkaline earth metal or a basic group containing a nitrogen orphosphorus atom.

For example, in the fluorescent group-containing compound of the presentinvention represented by the general formula (III), which can beobtained by allowing the fluorescent group-containing carbodiimidecompound precursor having the halogen atom or the sulfonic acid grouprepresented by the general formula (I) to react with the fluorescentgroup-containing carbodiimide compound represented by the generalformula (VIII), when at least one functional group selected from B, Y¹,Y², Y³, Y⁴, A and W derived from the precursor has at least one groupselected from a carboxyl group, a sulfo group, a phosphono group and aphospho group which have substitution of an alkali metal, an alkalineearth metal or a basic group containing a nitrogen or phosphorus atom,any of the functional groups represented by Y⁵, R⁴, R⁵ and F derivedfrom the fluorescent group-containing compound may or may not have agroup selected from a carboxyl group, a sulfo group, a phosphono groupand a phospho group which have substitution of an alkali metal, analkaline earth metal or a basic group containing a nitrogen orphosphorus atom.

Similarly, when at least one of functional groups represented by Y⁵, R⁴,R⁵ and F derived from the fluorescent group-containing compound has agroup selected from a carboxyl group, a sulfo group, a phosphono groupand a phospho group which have substitution of an alkali metal, analkaline earth metal or a basic group containing a nitrogen orphosphorus atom, any of the functional groups represented by B, Y¹, Y²,Y³, Y⁴, A and W derived from the precursor may or may not have a groupselected from a carboxyl group, a sulfo group, a phosphono group and aphospho group which have substitution of an alkali metal, an alkalineearth metal or a basic group containing a nitrogen or phosphorus atom.

Specific examples of the fluorescent group-containing carbodiimidecompound of the present invention represented by the general formula(III) include, for example,N-methyl-(3-morpholinopropyl)-N-(hexane-6-(N-3-(dimethylaminopropyl)-1-(RhodamineB)thiourea-potassium salt))-carbodiimide,N-methyl-(3-morpholinopropyl)-N-(hexane-6-(N-3-(dimethylaminopropyl)-1-(RhodamineB)thiourea-sodium salt))-carbodiimide,N-methyl-(3-morpholinopropyl)-N-(hexane-6-(N-3-(dimethylaminopropyl)-1-(RhodamineB)thiourea-tetramethylammonium salt))-carbodiimide,N-methyl-(3-morpholinopropyl)-N-(hexane-6-(N-3-(dimethylaminopropyl)-1-fluoresceinethiourea-potassiumsalt))-carbodiimide,2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-indol-2-ylidene]ethylidene]-2-phenoxy-1-cyclohexen-1-yl]-ethenyl]-1,1-dimethyl[3-propylthiourea-N-methyl-(3-morpholinopropyl)-N-(hexane-6-(N-3-(dimethylaminopropyl)-carbodiimide))]1H-benz[e]-indolium salt,N-methyl-(3-morpholinopropyl)-N-(hexane-6-(N-3-(dimethylaminopropyl)-1-(CascadeBlue)aminoethyl-4-azidobenzamide-trisodium salt))-carbodiimide and soforth.

The method for producing the fluorescent group-containing carbodiimidecompound of the present invention will be explained hereafter.

(2) Method for Producing Fluorescent Group-Containing CarbodiimideCompound

While the method for producing the fluorescent group-containingcarbodiimide compound of the present invention is not particularlylimited, it is generally produced by selecting a carbodiimide compoundand a fluorescent group-containing compound from the fluorescentgroup-containing carbodiimide compound precursor represented by thegeneral formula (I) and the fluorescent group-containing compoundrepresented by the general formula (VIII), respectively, so that atleast one of the carbodiimide compound precursor and the fluorescentgroup-containing compound should have a functional group selected from acarboxyl group, a sulfo group, a phosphono group and a phospho groupwhich have substitution of an alkali metal, an alkaline earth metal or abasic group containing a nitrogen or phosphorus atom, to allow them toreact with each other.

Specifically, the fluorescent group-containing carbodiimide compound ofthe present invention can be produced by performing (i) a step ofproducing the fluorescent group-containing carbodiimide compoundprecursor represented by the general formula (I), (ii) a step ofproducing the fluorescent group-containing compound represented by thegeneral formula (VIII), and (iii) a step of allowing the fluorescentgroup-containing carbodiimide compound precursor produced in the step(i) and the fluorescent group-containing compound produced in the step(ii) represented by the general formula (VIII) with each other. In theabove production, the fluorescent group-containing carbodiimide compoundprecursor represented by the general formula (I) and the fluorescentgroup-containing compound represented by the general formula (VIII) arecombined so that at least one of the both compounds should have afunctional group selected from a carboxyl group, a sulfo group, aphosphono group and a phospho group which have substitution of an alkalimetal, an alkaline earth metal or a basic group containing a nitrogen orphosphorus atom.

(i) Step of Producing Fluorescent Group-Containing Carbodiimide CompoundPrecursor Represented by the Formula (I)

The fluorescent group-containing carbodiimide compound precursor havingthe halogen atom or the sulfonic acid group used for the presentinvention can be suitably selected according to a structure of a desiredfluorescent group-containing carbodiimide compound. The method forproducing such a fluorescent group-containing carbodiimidegroup-containing compound precursor having a halogen atom or a sulfonicacid group is not particularly limited. For example, it can be producedby (A) preparation of a urea or thiourea compound starting from anisocyanate compound and an amine compound such as a primary aminederivative having a functional group required for conversion into ahalogenated derivative or sulfonated derivative, (B) halogenation orsulfonation of the (thio)urea compound, and (C) carbodiimidation of theresulting compound by dehydration or oxidative desulfurization. Thesteps of (A) to (C) will be explained in detail hereinafter.

(A) Step of Producing (thio)urea Compound

Urea compounds can generally be synthesized by a reaction of an aminecompound and an isocyanate compound [J. H. Saunders and R. Slocombe,Chem. Rev., 43, 203 (1948)]. Further, it is a common practice thatthiourea derivatives are produced by a reaction of an amine compound andan isothiocyanate compound [N. A. Ivanov, R. V. Viasova, V. A.Gancharava, and L. N. Smirnov, Izv. Vyssh. Zaved. Khim. Tekhnol., 19(7), 1010 (1976)].

The aforementioned methods can also be used for the production method ofthe present invention. The (thio)urea compound represented by thegeneral formula (VI) can be synthesized by allowing the iso(thio)cyanatecompound represented by the general formula (IV) and an amine compoundwhich has a functional group required for the conversion into ahalogenated derivative or sulfonated derivative represented by thegeneral formula (V) to react with each other. The (thio)urea compoundrepresented by the general formula (VI) obtained by this reaction alsohas a functional group required for the conversion into a halogenatedderivative or sulfonated derivative, for example, a hydroxyl group, analkyl group, an alkenyl group, a vinyl group, an allyl group, a phenylgroup, a carboxyl group, a sulfonyl group-containing derivative group, aphosphonium group and so forth.

As specific examples of the aforementioned reaction, there can bementioned, for example, a reaction by mixing the iso(thio)cyanatecompound represented by the general formula (IV), such asN-(3-morpholinopropyl)isothiocyanate,N-3-dimethylaminopropyl-N-3-(4-morpholino)-propyl isothiocyanate,1-ethyl-3,3-dimethylaminopropyl isothiocyanate andbis(3,3-dimethylamino)propyl isothiocyanate, with the amine compoundhaving the functional group required for the conversion into thehalogenated derivative or sulfonated derivative represented by thegeneral formula (V), such as 6-aminohexanol, 5-aminopentanol,4-aminobutanol and 3-aminopentanol, in a known solvent such asdichloromethane, chloroform and dimethylformamide (abbreviated as “DMF”hereinafter). Thus, the (thio)urea compound having the functional grouprequired for the conversion into the halogenated derivative orsulfonated derivative represented by the general formula (VI) can beobtained.

Moreover, besides the aforementioned method, a first amine compound andurea can be condensed to obtain a monosubstituted urea, and themonosubstituted urea can be reacted with a second primary amine having afunctional group required for the conversion into a halogenatedderivative or sulfonated derivative to obtain a disubstituted ureaintermediate [T. L. Davis and K. C. Blanchard, Org. Synth. Coll Vol. 1,453 (1941)] as the following reaction scheme:

wherein, B, Y¹, Y², Y³, Y⁴, A, W and n have the same meanings as definedfor the general formula (I); T represents an oxygen atom or a sulfuratom; and Z represents a hydroxyl group, an alkyl group, an alkenylgroup, a vinyl group, an allyl group, a phenyl group, a carboxyl group,a sulfonyl group-containing derivative group or a phosphonium group.

Moreover, the monosubstituted urea can also be synthesized by a reactionof the first amine compound and cyanic acid or a salt thereof [F. KurzerOrg. Synth. Coll. Vol. 4, 49 (1963)]. Furthermore, it is also possibleto use a primary amine having a functional group required for theconversion into a halogenated derivative or sulfonated derivative in theabove reactions. In this case, the second amine compound may or may nothave a functional group required for the conversion into a halogenatedderivative or sulfonated derivative.

In addition, the disubstituted urea derivative can also be directlyobtained by a reaction of an amine compound having a functional grouprequired for the conversion into a halogenated derivative or sulfonatedderivative and an isocyanate compound.

When the (thio)urea compound represented by the general formula (VI) hasa tertiary nitrogen atom or phosphorus atom-containing group, such as amorpholinoalkyl group, a morpholinoalkyl ester group, a morpholinoalkylether group, a morpholinoalkylamide group, a morpholinoalkyl ketonegroup, a morpholinoalkylthiourea group, a trialkylamino group, a(trialkyl ester)amino group, a trialkyl ether group, atrialkylamidoamino group, a (trialkyl ketone)amino group, atrialkylthioureamino group, a trialkylphosphonium group, a (trialkylester)phosphonium group, a (trialkyl ether)phosphonium group, atrialkylamidophosphonium group, a (trialkyl ketone)phosphonium group,and a trialkylthioureaphosphonium group, its water solubility may beimproved by making it into a quaternary onium salt through a reactionwith methyl p-toluenesulfonate, methyl iodide, dimethyl sulfate and soforth in a known solvent such as DMF.

As explained below, the aforementioned step (A) can also be performed bytwo stages of (A′) a step of producing a urea or thiourea compoundstarting from an iso(thio)cyanate compound and an amine compound such asprimary amine derivatives, and (A″) a subsequent step of introducing afunctional group required for conversion into a halogenated derivativeor sulfonated derivative into the (thio)urea compound.

In this method, a (thio)urea compound represented by the followinggeneral formula (IV′) is synthesized first in the step (A′) through areaction of an iso(thio)cyanate compound similar to that represented bythe general formula (IV) and an amine compound represented by thefollowing general formula (V′). Like the aforementioned reaction, thisreaction is also a common reaction of an iso(thio)cyanate compound andan amine compound.

In the formulae, B, Y² and Y³ have the same meanings as defined for thegeneral formula (I); W′ represents a group containing secondary ortertiary nitrogen atom or secondary or tertiary phosphorus atom, whichmay have substitution of a group selected from a hydroxyl group, analkyl group, an alkenyl group, a vinyl group, an allyl group, a phenylgroup, a carboxyl group, a sulfonyl group-containing derivative groupand a phosphonium group; and T represents an oxygen atom or a sulfuratom.

Such a thiourea compound as mentioned below can also be synthesized asthe compound represented by the formula (IV′) through a reaction of anamine compound and carbon disulfide [W. W. Levis, Jr. and E. A. Waipert,U.S. Pat. No. 3,168,560 (1965)].

In the formulae, B and Y³ have the same meanings as defined for thegeneral formula (I).

In addition, such a disubstituted urea intermediate as mentioned belowcan be synthesized as the compound represented by the formula (IV′) bycondensing an amine compound and urea to obtain a monosubstituted urea,and allowing the product to react with a second amine or the same amine[T. L. Davis and K. C. Blanchard, Org. Synth. Coll. Vol. 1, 453 (1941)].

In the formulae, B, Y² and Y³ have the same meanings as defined for thegeneral formula (I); and W′ represents an onium group which may havesubstitution.

Moreover, the monosubstituted urea can also be synthesized through areaction of an amine compound and cyanic acid or a salt thereof [F.Kurzer, Org. Synth. Coll. Vol. 4, 49 (1963)].

Then, the step (A″) is performed, wherein the (thio)urea compoundrepresented by the general formula (IV′) obtained in the aforementionedstep (A′) is allowed to react with an amine compound having a functionalgroup required for the conversion into a halogenated derivative orsulfonated derivative represented by the general formula (V″) to producethe compound represented by the general formula (VI).

In the formulae, B, Y¹, Y², Y³, Y⁴, W, A and n have the same meanings asdefined for the general formula (I); T represents an oxygen atom or asulfur atom; D represents a hydroxyl group, a halogen atom or a sulfonylgroup-containing derivative group; and Z represents a hydroxyl group, analkyl group, an alkenyl group, a vinyl group, an allyl group, a phenylgroup, a carboxyl group, a sulfonyl group-containing derivative group ora phosphonium group.

As described above, the (thio)urea compound represented by the generalformula (VI), which has a functional group required for the conversioninto a halogenated derivative or sulfonated derivative, can be obtainedin the step (A), through the aforementioned one-step reaction process,or the two-step reaction process of the steps (A′) and (A″).

(B) Step of Halogenation or Sulfonation

As to the method for introducing a halogen atom into the (thio)ureacompound represented by the general formula (VI), when a hydroxyl grouphas been introduced into the (thio)urea compound, the halogen atom canbe introduced into the (thio)urea compound by, for example, mixing the(thio)urea compound with a halogenation reagent, for example, a hydrogenhalide, a sodium halide, a potassium halide, a phosphorus halide,halogenated phosphonic acid triester, a phosphine halide, a thionylhalide, an acid halogen compound and so forth in a known solvent such asDMF, benzene and pyridine to allow them to react with each other [O.Kamm C. S. Marvel, Org. Syhth., 1, 25 (1941); T. A. Wnuk, P. Kovacic, J.Am. Chem. Soc., 97, 5807 (1975); J. D. Bartleson, R. E. Burk, H. P.Lankelma, J. Am. Chem. Soc., 68, 2513 (1946); K. Friedlich, H. K.Thieme, Synthesis, 111(1973); H. Stone, H. Shechter, Org. Synth., 4, 323(1963); T. H. Bevan, T. Malkin, D. B. Smith, J. Chem. Soc., 1383(1955)].

Specific examples of the halogenation reagent include hydrobromic acid,sodium bromide, zinc chloride, phosphorus tribromide, phosphorustrichloride, potassium iodide, methyltriphenylphosphonic acid iodide,iodine, sodium iodide, methanesulfonyl chloride, triphenylphosphinedibromide, triphenylphosphine dichloride, triphenylphosphine diiodide,thionyl chloride, thionyl bromide and so forth.

Further, as to the method for introducing a sulfonic acid group into the(thio)urea compound represented by the general formula (VI), when ahydroxyl group has been introduced into the (thio)urea compound, thesulfonic acid group can be introduced into the (thio)urea compound by,for example, mixing the compound with a sulfonation reagent such as asulfonic acid halide in a known solvent such as dichloromethane andchloroform to allow them to react with each other.

Specific examples of the sulfonation reagent include benzenesulfonicacid chloride, p-toluenesulfonic acid chloride, methanesulfonic acidchloride, benzenesulfonic acid bromide, p-toluenesulfonic acid bromide,methanesulfonic acid bromide and so forth.

Thus, a halogenated or sulfonated derivative of the (thio)urea compoundrepresented by the general formula (VII) can be obtained by halogenatingor sulfonating the (thio)urea compound represented by the generalformula (VI).

(C) Step of Dehydration or Oxidative Desulfurization

In the production method of the present invention, the fluorescentgroup-containing carbodiimide compound precursor represented by thegeneral formula (I) of the present invention can be derived from thecompound represented by the general formula (VII) by subjecting it todehydration or oxidative desulfurization reaction.

The dehydration reaction is used for the urea compound containing anoxygen atom as T among the compounds represented by the general formula(VII), and it can be performed by heating the urea compound withp-toluenesulfonic acid chloride in a tertiary amine compound [G. Amiardand R. Heymers, Bull. Soc. Chim. Fr., 1360 (1956)]. Further, it can alsobe performed by using p-toluenesulfonic acid chloride and potassiumcarbonate in the presence of a quaternary ammonium salt [Zsuzsa M.Jaszay., Synthesis, 520 (1987)].

As for the thiourea compounds containing a sulfur atom as T among thecompounds represented by the general formula (VII), oxidativedesulfurization reaction is performed in order to obtain the fluorescentgroup-containing carbodiimide compound precursor of the presentinvention. In general, mercury oxide is used for the oxidativedesulfurization reaction as a desulfurization agent. Besides mercuryoxide, lead oxide [F. Zetzehe and A. Fredrich, Chem. Ber., 73, 1114(1940)], zinc oxide [R. F. Coles, U.S. Pat. No. 2,946,819 (1960)], leadcarbonate, lead nitrate, lead chloride [J. C. Sheehan, U.S. Pat. No.3,135,748 (1964)) and so forth can also be used. Furthermore, sodiumhypochlorite may also be used under an alkaline condition [H. Stetterand C. Wulff, Chem. Ber., 95, 2302 (1962)].

Although the fluorescent group-containing carbodiimide compoundprecursor of the present invention having the halogen atom or thesulfonic acid group, which is represented by the general formula (I),can be produced as described above, the aforementioned production methodis a mere example, and the order of the process steps is not limited tothat mentioned above. For example, either of the halogenation orsulfonation and the carbodiimidation may precede the other process step.

Further, when the step (A) is performed by a reaction process of twosteps, steps (A′) and (A″), it is also possible to produce thefluorescent group-containing carbodiimide compound precursor of thepresent invention having the halogen atom or the sulfonic acid group,which is represented by the general formula (I), by carbodiimidating thecompound represented by the general formula (VI′) obtained in the step(A′), introducing, into the compound, a functional group required forthe conversion into a halogen derivative or a sulfone derivative in thestep (A″), and finally subjecting the compound to the halogenation step.

Thus, there is obtained the fluorescent group-containing carbodiimidecompound precursor of the present invention having the halogen atom orthe sulfonic acid group, which is represented by the general formula(I). In the general formula (I), a group selected from a carboxyl group,a sulfo group, a phosphono group and a phospho group which havesubstitution of an alkali metal, an alkaline earth metal or a basicgroup containing a nitrogen or phosphorus atom replaceable hydrogenatoms may be substituted for any replaceable hydrogen atom present onany of the functional groups represented by B, Y¹, Y², Y³, Y⁴, A and W.

The aforementioned group selected from a carboxyl group, a sulfo group,a phosphono group and a phospho group which have substitution of analkali metal, an alkaline earth metal or a basic group containing anitrogen or phosphorus atom may be contained in a raw material compoundused for the production of the fluorescent group-containing carbodiimidecompound precursor represented by the general formula (I) describedabove, or it may be introduced into the raw material compound at thefirst stage of the production, or it may be introduced into anintermediate compound during the production, when the raw materialcompound does not contain the group. Furthermore, when a finallyobtained fluorescent group-containing carbodiimide compound precursorrepresented by the general formula (I) does not contain such a group, itmay further be introduced into the final compound.

In order to introduce the group, for example, a compound having a groupselected from a carboxyl group, a sulfo group, a phosphono group and aphospho group, and a functional group for the introduction of the groupcan be made into an alkali metal salt such as potassium salt, analkaline earth metal salt such as calcium and magnesium salt, or a saltwith a basic group containing a nitrogen or phosphorus atom at thecarboxyl group, the sulfo group, the phosphono group or the phosphogroup, and introduced into a raw material or an intermediate of thefluorescent group-containing carbodiimide compound precursor, or thefinal product. Alternatively, the compound having a carboxyl group, asulfo group, a phosphono group, a phospho group or the like not madeinto a salt can also be introduced into the raw material or theintermediate of the precursor, or the final product, and then thefunctional groups may be converted into such salts as mentioned above.

When the raw material of the precursor is a compound having a groupselected from a carboxyl group, a sulfo group, a phosphono group and aphospho group, it may be converted into an alkali metal salt, analkaline earth metal salt, or a salt of a basic group containing anitrogen or phosphorus atom, and then used for the production process.Alternatively, the carboxyl group, the sulfo group, the phosphono groupand the phospho group of the raw material may not be converted into asalt, and the raw material may be used as it is for the productionprocess, and those functional groups of a compound obtained after anappropriate step can be converted into a salt.

The method for converting the aforementioned compound into an alkalimetal salt, an alkaline earth metal salt or a salt of a basic groupcontaining a nitrogen or phosphorus atom salt at the carboxyl group, thesulfo group, the phosphono group or the phospho group possessed by thecompound is not particularly limited. Any known methods can be used forit.

Specifically, a method comprising adding a known solvent such as waterand alcohol to the compound having a group selected from a carboxylgroup, a sulfo group, a phosphono group and a phospho group, to dissolvethe compound in the solvent, and then adding to the obtained solution,sodium hydroxide, potassium hydroxide, calcium hydroxide, sodiumcarbonate, potassium carbonate, sodium hydrogencarbonate,tetramethylammonium hydroxide, tetramethylammonium chloride,tetramethylammonium bromide, tetramethylammonium iodide or the like, andmixing to allow to cause reaction.

(ii) Step of Producing Fluorescent Group-Containing Compound Representedby the General Formula (VIII)

The fluorescent group-containing compound represented by the generalformula (VIII) which is usable for producing the fluorescentgroup-containing carbodiimide compound of the present invention that hasa group selected from a carboxyl group, a sulfo group, a phosphono groupand a phospho group which have substitution of an alkali metal, analkaline earth metal or a basic group containing a nitrogen orphosphorus atom, by allowing the fluorescent group-containing compoundrepresented by the general formula (VIII) to react with the fluorescentgroup-containing carbodiimide compound precursor having the halogen atomor the sulfonic acid group obtained in the aforementioned (i), can beselected depending on desired fluoresent group-containing carbodiimidecompounds.

In order to obtain the fluorescent group-containing compound representedby the general formula (VIII), a group containing a secondary ortertiary nitrogen atom or a secondary or tertiary phosphorus atom can beintroduced into a suitable fluorescent group-containing compoundcontaining a fluorescent group F according to a usual method. Examplesof the fluorescent group-containing compound include anthracenederivatives, coumarin derivatives, pyrene derivatives, perylenederivatives, dansyl derivatives, rhodamine derivatives, oxazolederivatives, benzothiazole derivatives, benzoxadiazole derivatives,boron dipyrromethene difluoride derivatives, thiazole orangederivatives, fluorescent rare earth metal compound derivatives, cyaninederivatives and so forth. The method for producing the fluorescentgroup-containing compound represented by the general formula (VIII),which contains the fluorescent group F and the group containing thesecondary or tertiary nitrogen atom or the secondary or tertiaryphosphorus atom, is not particularly limited.

As a specific example, a method for producing a compound having—(CH₂)_(p)—NHC(S)NH—(CH₂)_(q)— as Y⁵ in the general formula (VIII) isshown below. This reaction is a usual method comprising a reaction of acompound containing an isothiocyanate group and a fluorescent group, andan amine compound having a secondary or tertiary nitrogen or phosphorusatom.

In the formulae, R⁴, R⁵ and F have the same meanings as defined for theformula (III); and Q′ has the same meaning as defined for the formula(VIII).

As further specific examples, there can be mentioned methods comprisingmixing fluorescein isothiocyanate, Rhodamine B isothiocyanate,2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-indol-2-ylidene]ethylidene]-2-phenoxy-1-cyclohexen-1-yl]-ethenyl]-1,1-dimethyl[3-propylisothiocyanate] 1H-benz[e]-indolium salt or the like with an aminederivative having a primary or tertiary amino group such asN-3-dimethylaminopropyl-N-3-(4-morpholino)propylene,1-ethyl-3,3-dimethylaminopropylene, bis-(3,3-dimethyl)aminopropylene andbis(3,3-dimethylamino)propylene in a known solvent such asdichloromethane, chloroform and DMF, to react them each other.

As described above, the fluorescent group-containing compoundrepresented by the general formula (VIII) can be obtained. A carboxylgroup, a sulfo group, a phosphono group and a phospho group which havesubstitution of an alkali metal, an alkaline earth metal or a basicgroup containing a nitrogen or phosphorus atom may be substituted forreplaceable hydrogen atoms present in any of the functional groupsrepresented by Y⁵, R⁴, R⁵ and F in the general formula (VIII).

In the production of the fluorescent group-containing compoundrepresented by the general formula (VIII), the group selected from thecarboxyl group, the sulfo group, the phosphono group and the phosphogroup which have substitution of the alkali metal, the alkaline earthmetal or the basic group containing the nitrogen or phosphorus atom maybe contained in a raw material, or introduced into a raw material in thefirst stage of the production when the raw material does not contain thegroup, or introduced into an intermediate during the production.Furthermore, when the fluorescent group-containing compound representedby the general formula (VIII) finally obtained does not contain such agroup, the group may be introduced into the compound after itsproduction.

The introduction of the group may be attained in the same manner as thecase of the aforementioned fluorescent group-containing carbodiimidecompound precursor. When the raw material contains a group selected froma carboxyl group, a sulfo group, a phosphono group and a phospho group,it can be treated in a manner similar to the case of the aforementionedfluorescent group-containing carbodiimide compound precursor. Further, acompound having a group selected from a carboxyl group, a sulfo group, aphosphono group and a phospho group can be converted into a salt with analkali metal, an alkaline earth metal or a basic group containing anitrogen or phosphorus atom at those functional groups in the samemanner as described above.

(iii) Step of Allowing Fluorescent Group-Containing CarbodiimideCompound Precursor Produced in Step (i) and Fluorescent Group-ContainingCompound Represented by the General Formula (VIII) Produced in the Step(ii) to React with Each Other

The fluorescent group-containing carbodiimide compound of the presentinvention having at least one group selected from a carboxyl group, asulfo group, a phosphono group and a phospho group which havesubstitution of an alkali metal, an alkaline earth metal or a basicgroup containing a nitrogen or phosphorus atom, which is represented bythe general formula (III), can be produced by allowing the fluorescentgroup-containing carbodiimide compound precursor represented by thegeneral formula (I) produced in step (i) and the fluorescentgroup-containing compound represented by the general formula (VIII)produced in the step (iii) to react with each other.

At least one of the fluorescent group-containing carbodiimide compoundprecursor having the halogen atom or the sulfonic acid group representedby the general formula (I) and the fluorescent group-containing compoundrepresented by the general formula (VIII) must necessarily be a compoundcontaining at least one group selected from a carboxyl group, a sulfogroup, a phosphono group and a phospho group which have substitution ofan alkali metal, an alkaline earth metal or a basic group containing anitrogen or phosphorus atom.

That is, when the fluorescent group-containing carbodiimide compoundprecursor having the halogen atom or the sulfonic acid group has atleast one group selected from a carboxyl group, a sulfo group, aphosphono group and a phospho group which have substitution of an alkalimetal, an alkaline earth metal or a basic group containing a nitrogen orphosphorus atom, i.e., when at least one functional group selected fromB, Y¹, Y², Y³, Y⁴, A and W in the general formula (I) has at least onegroup selected from a carboxyl group, a sulfo group, a phosphono groupand a phospho group which have substitution of an alkali metal, analkaline earth metal or a basic group containing a nitrogen orphosphorus atom, the fluorescent group-containing compound representedby the general formula (VIII) may or may not have the aforementionedfunctional group.

Similarly, when the fluorescent group-containing compound represented bythe general formula (VIII) has at least one group selected from acarboxyl group, a sulfo group, a phosphono group and a phospho groupwhich have substitution of an alkali metal, an alkaline earth metal or abasic group containing a nitrogen or phosphorus atom, i.e., when atleast one functional group selected from Y⁵, R⁴, R⁵ and F in the generalformula (VIII) has at least one group selected from a carboxyl group, asulfo group, a phosphono group and a phospho group which havesubstitution of an alkali metal, an alkaline earth metal or a basicgroup containing a nitrogen or phosphorus atom, the fluorescentgroup-containing carbodiimide compound precursor having the halogen atomor the sulfonic acid group represented by the formula (I) may or may nothave the aforementioned functional group.

The method for such a reaction of the fluorescent group-containingcarbodiimide compound precursor having the halogen atom or the sulfonicacid group represented by the general formula (I) and the fluorescentgroup-containing compound represented by the general formula (VIII) asmentioned above is not particularly limited. Specifically, theproduction can be attained by mixing the both compounds in anappropriate ratio in a known solvent such as dichloromethane, chloroformand DMF, to allow them to react with each other.

When the fluorescent group-containing carbodiimide compound of thepresent invention is produced, for example, even if the fluorescentgroup-containing compound has a functional group such as a carboxylgroup, a sulfo group, a phosphono group and a phospho group in itsstructure, a carbodiimide group can be introduced without removing thefunctional group from the compound beforehand and with leaving it in theform of a salt, by using the fluorescent group-containing carbodiimidecompound precursor which has the halogen or the sulfonic acid group,which is represented by the general formula (I). Thus, it enables theproduction with fewer process steps as compared with conventionalmethods.

Moreover, a carbodiimide group can easily be introduced into acommercially available fluorescent group-containing compound containinga functional group such as a carboxyl group, a sulfo group, a phosphonogroup and a phospho group in its structure, by converting the functionalgroup into a salt. Therefore, kinds of usable fluorescentgroup-containing compounds can be extremely increased.

The fluorescent group-containing carbodiimide compound of the presentinvention obtained by the method described above can be suitably used asa labeling substance in methods for detecting nucleic acids orimmunoassay methods. In these applications, the fluorescentgroup-containing carbodiimide compound of the present invention can bebonded to a nucleic acid such as DNA or a protein such as antigen and anantibody to be labeled by bringing the fluorescent group-containingcarbodiimide compound into contact with the nucleic acid or the proteinby, for example, mixing them in a solvent. That is, by bonding thefluorescent group-containing carbodiimide compound of the presentinvention to a nucleic acid or a protein via the carbodiimide group,which is highly reactive with nucleic acid bases, a fluorescentsubstance functioning as a highly sensitive detection reagent can beadded to the nucleic acid or the protein to serve as a label. Therefore,when the fluorescent group-containing carbodiimide compound of thepresent invention is bonded to a nucleic acid or a protein, it ispreferable that the both should be brought into contact under acondition that allows easy reaction of the carbodiimide group, forexample, an alkaline condition of about pH 7.5 to about pH 8.5.Furthermore, the fluorescent group-containing carbodiimide compound ofthe present invention can also be used for chemiluminescence analysismethods and so forth.

Moreover, in the fluorescent group-containing carbodiimide compound ofthe present invention, the carbodiimide group can exist in one systemtogether with salts of functional groups such as a carboxyl group, asulfo group, a phosphono group and a phospho group. Therefore, it hassufficiently improved in water-solubility compared with conventionalfluorescent group-containing carbodiimide compounds, and it isadvantageous when it is used as a labeling substance in theaforementioned methods for detecting nucleic acids or immunoassaymethods as well as chemiluminescence analysis methods and so forth.

(3) Method for Detecting Nucleic Acid of the Present Invention

The fluorescent group-containing carbodiimide compound of the presentinvention having at least one group selected from the carboxyl group,the sulfo group, the phosphono group and the phospho group which havesubstitution of the alkali metal, the alkaline earth metal or the basicgroup containing the nitrogen or phosphorus atom can be used as alabeling substance in a method for detecting a nucleic acid byhybridization using a nucleic acid labeled with a labeling substance.That is, a nucleic acid labeled with the fluorescent group-containingcarbodiimide compound can be used as a probe for hybridization. Anucleic acid to be analyzed can be detected by hybridizing the probewith the nucleic acid to be analyzed so that a nucleic acid/nucleic acidhybrid should be formed, eliminating any free probe from the system, anddetecting the labeling substance contained in the hybrid. According tothe present invention, the fluorescent group-containing carbodiimidecompound serving as a labeling substance can be directly detected bymeasuring fluorescence intensity or the like by using aspectrophotofluorometer, a spectrophotofluorometer for a 96-wellmicrotiter plate, a fluorescence microscope and so forth. The nucleicacid to be measured is usually used as fixed on a membrane such as nylonmembranes and cellulose nitrate membranes or a microtiter plate.

The hybridization used in the method for detecting the nucleic acid ofthe present invention does not particularly differ from usualhybridization of nucleic acids such as colony hybridization, plaquehybridization, dot blot hybridization, Southern hybridization andNorthern hybridization, except that the hybridization of the method fordetecting the nucleic acid of the present invention uses the fluorescentgroup-containing carbodiimide compound for the label of a nucleic acidprobe. The nucleic acid to be measured may be either RNA or DNA, and thenucleic acid used for a probe may also be either RNA or DNA.

While the labeling of a nucleic acid used for a probe is preferablyperformed by labeling a polynucleotide or oligonucleotide by the methoddescribed above, it can also be attained by incorporating a labelednucleotide into a polynucleotide or oligonucleotide through a polymerasereaction.

EXAMPLES

Hereafter, the present invention will be explained with reference to thefollowing examples.

Example 1

(1) Production of Fluorescent Group-Containing Carbodiimide CompoundPrecursor Represented by the General Formula (I)

N-(3-Morpholinopropyl) isothiocyanate (1.68 g, 10 mmol) was dissolved in15 ml of dry methylene chloride, and cooled on a water bath. To thesolution, 6-aminohexanol (1.17 g, 10 mmol) was added, and the mixturewas stirred overnight at room temperature. After addition of water tothe reaction mixture, the mixture was extracted with methylene chloride.The extract was dried over anhydrous magnesium sulfate, filtered throughCelite, and concentrated to obtain Compound (I-I-A) (2.7 g, yield: 95%).The NMR spectrum data are shown below.

¹H-NMR (CDCl₃): δ=1.35–1.85 (m, 12H), δ=2.40–2.60 (m, 6H), δ=3.45 (br,2H), δ=3.65 (t, 2H), δ=3.75 (t, 4H)

Then, the obtained Compound (I-I-A) (2.0 g, 4.87 mmol) was dissolved indry DMF (5 ml). Methyl iodide (0.691 g, 4.87 mmol) was added to thesolution, and the mixture was stirred overnight at room temperature.After the reaction mixture was concentrated, ether/methanol was addedthereto and the mixture was decanted to obtain Compound (I-I-B) (2.6 g,yield: 97%). The NMR spectrum data are shown below.

¹H-NMR (DMSO): δ=1.25–1.65 (m, 10H), δ=1.75 (t, 2H), δ=2.30–2.45 (m,2H), δ=2.65 (s, 3H), δ=3.20–3.50 (m, 6H), δ=3.60 (t, 4H), δ=3.75 (br,2H)

Then, the obtained compound (I-I-B) (2 g, 3.62 mmol) was dissolved inDMF (5 ml). Methyl-(triphenyl)-phosphonium iodide (3.27 g, 7.23 mmol)was added thereto, and the mixture was stirred at room temperature for 3hours. Then, methanol (5 ml) was added thereto, and the mixture wasstirred for 20 minutes. Further, this reaction mixture was concentrated,and purified by silica gel chromatography (developing solvent:chloroform/methanol=50/1) to isolate Compound (I-I-C) (2.98 g, yield:95%). The NMR spectrum data are shown below.

¹H-NMR (CDCl₃): δ=1.30–1.90 (m, 10H), δ=2.25 (t, 2H), δ=2.85–3.00 (m,6H), δ=2.95 (s, 3H), δ=3.20 (t, 2H), δ=3.45 (br, 2H), δ=3.90 (t, 4H)

Then, the obtained Compound (I-I-C) (2 g, 2.31 mmol) was dissolved inacetone (5 ml). Zinc oxide (1 g, 4.62 mmol) was added thereto, and themixture was stirred for 3 hours under reflux. The reaction mixture wascooled by leaving it for 1 hour, then decanted, and concentrated toobtain Compound (I-I-D) (1.73 g, yield: 90%). This compound is one ofthe fluorescent group-containing carbodiimide compound precursors of thepresent invention. The NMR spectrum data and IR spectrum data are shownbelow.

¹H-NMR (CDCl₃): δ=1.30–1.90 (m, 10H), δ=2.40–2.50 (m, 8H), δ=3.15 (s,3H), δ=3.20 (t, 2H), δ=3.30 (t, 4H), δ=3.75 (t, 4H). IR: 2127 cm⁻¹(—N═C═N— group)

(2) Production of Fluorescent Group-Containing Compound Represented bythe General Formula (VIII)

Rhodamine B isothiocyanate (2.00 g, 3.73 mmol, produced by Sigma) wasdissolved in dichloromethane. Ater this reaction mixture was cooled to0° C., N-3-(dimethylaminopropyl)amine (0.57 g, 5.60 mmol) was graduallyadded thereto, and the mixture was stirred for 30 minutes. After thisreaction mixture was concentrated, ether/methanol was further addedthereto, and the mixture was decanted to obtain Compound (I-II-A) (2.26g, yield: 95%). The NMR spectrum data are shown below.

¹H-NMR (CDCl₃): δ=1.00–1.20 (m, 8H), δ=2.20–2.60 (m, 6H), δ=2.20 (s,6H), δ=3.25–3.70 (m, 4H), δ=6.30–8.00 (m, 9H)

The obtained Compound (I-II-A) (2.26 g, 3.54 mmol) was dissolved inmethanol. A saturated solution of potassium hydroxide in isopropylalcohol (about 15 ml) was added thereto to allow deposition of solidmatter. Then, it was filtered by using ethanol, and dried to obtainCompound (I-II-B) (2.30 g, yield: 96%). This compound corresponded tothe compound (I-II-A) in which the carboxyl group in the Rhodamine Bgroup was made into a potassium salt (—COOK). The NMR spectrum data areshown below.

¹H-NMR (CDCl₃): δ=1.00–1.20 (m, 8H), δ=2.20–2.60 (m, 6H), δ=2.20 (s,6H), δ=3.25–3.70 (m, 4H), δ=6.30–8.00 (m, 9H).

(3) Production of Fluorescent Group-Containing Carbodiimide CompoundRepresented by the General Formula (III)

Compound (I-I-D) obtained in (1) (1.23 g, 1.48 mmol) and Compound(I-II-B) obtained in (2) (1 g, 1.48 mmol) were dissolved in DMF, and thesolution was stirred overnight at room temperature. To the reactionmixture, ether/methanol was added and the mixture was decanted to obtainCompound (1) (2.10 g, yield: 94%) as the fluorescent group-containingcarbodiimide compound of the present invention. The NMR spectrum data,IR spectrum data, and UV spectrum data are shown below.

¹H-NMR (CDCl₃): δ=1.00–1.85 (m, 18H), δ=2.40–2.55 (m, 12H), δ=2.85 (s,3H), δ=2.90 (s, 6H), δ=3.15 (t, 2H), δ=3.25 (t, 4H), δ=3.50–3.65 (m,6H), δ=3.75 (t, 4H), δ=6.30–8.00 (m, 9H). IR: 2125 cm⁻¹ (—N═C═N— group)UV (EtOH): λ_(max) 542 nm

Example 2

(1) Production of Fluorescent Group-Containing Compound Represented bythe General Formula (VIII)

2-[2-[3-[[1,3-Dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-indol-2-ylidene]ethylidene]-2-phenoxy-1-cyclohexen-1-yl]-ethenyl]-1,1-dimethyl[3-propylisothiocyanate] 1H-benz [e]-indolium salt (400 mg, 0.502 mmol, producedby LI-COR) was dissolved in dichloromethane (10 ml). After the solutionwas cooled to 0° C., N-3-(dimethylaminopropyl)amine (76.9 mg, 0.753mmol) was gradually added thereto, and the mixture was stirred at roomtemperature for 1.5 hours. After this reaction solution wasconcentrated, ether/methanol was added, and the mixture was decanted toobtain Compound (II-I-A) (402 mg, yield: 89%). The NMR spectrum data areshown below.

¹H-NMR (CDCl₃): δ=1.20–2.40 (m, 14H), δ=2.25 (s, 6H), δ=2.45 (t, 2H),δ=2.65–3.05 (m, 4H), δ=3.65–3.95 (m, 4H), δ=4.45 (t, 2H), δ=6.13 (d,1H), δ=6.35 (d, 1H), δ=6.90–7.60 (m, 11H), δ=7.70–8.15 (m, 5H)

(2) Production of Fluorescent Group-Containing Carbodiimide CompoundRepresented by the General Formula (III)

Compound (I-I-D) obtained in the Example 1 (1) (232 mg, 0.445 mmol) andCompound (II-I-A) obtained in the Example 2 (1) (400 mg, 0.445 mmol)were dissolved in DMF (10 ml), and the solution was stirred overnight atroom temperature. To the reaction mixture, ether/methanol was furtheradded and the mixture was decanted to obtain Compound (2) (575 mg,yield: 91%) as the fluorescent group-containing carbodiimide compound ofthe present invention. The NMR spectrum data, IR spectrum data, and UVspectrum data are shown below.

¹H-NMR (CDCl₃): δ=1.20–2.10 (m, 24H), δ=2.30–2.50 (m, 12H), δ=3.10–3.35(m, 4H), δ=2.90 (s, 3H), δ=2.95 (s, 6H), δ=3.60–3.85 (m, 10H), δ=6.13(d, 1H), δ=6.35 (d, 1H), δ=6.80–7.60 (m, 11H), δ=7.80–8.18 (m, 5H) IR:2127 cm⁻¹ (—N═C═N— group) UV (EtOH): λ_(max) 790 nm

Example 3

(1) Production of Fluorescent Group-Containing Compound Represented bythe General Formula (VIII)

Cy3.18.OSu (200 mg, 0.175 mmol) produced by referring to Japanese PatentApplication Laid-open No. 9-325147 and Ratnakaer B. Mujumdar et. al.,Bioconjugate Chem., 4, 2, 105–111 (1993) was dissolved in DMF (5 ml).After the reaction solution was cooled to 0° C.,N-3-(dimethylaminopropyl)amine (71.4 mg, 0.698 mmol) was gradually addedthereto, and the mixture was stirred at room temperature for 12 hours.After this reaction solution was concentrated, ethyl acetate/methanolwas added thereto, and the mixture was decanted several times to obtainCompound (III-I-A) (170 mg, yield: 87%). The NMR spectrum data are shownbelow.

¹H-NMR (DMSO): δ=1.30–1.60 (m, 12H), δ=1.70 (s, 12H), δ=2.05 (t, 4H),δ=2.10–2.30 (m, 8H), δ=2.15 (s, 12H), δ=2.95 (q, 4H), δ=4.12 (t, 4H),δ=6.54 (d, 2H), δ=7.36–7.95 (m, 6H), δ=8.36 (t, 1H).

(2) Production of Fluorescent Group-Containing Carbodiimide CompoundRepresented by the General Formula (III)

Compound (I-I-D) (79 mg, 0.152 mmol) obtained in the Example 1 (1) andCompound (III-I-A) (170 mg, 0.152 mmol) obtained in the Example 3 (1)were dissolved in DMF (10 ml), and the solution was stirred overnight atroom temperature. To the reaction mixture, ethyl acetate/methanol wasfurthere added and the mixture was decanted to obtain Compound (3) (249mg, yield: 100%) as the fluorescent group-containing carbodiimidecompound of the present invention. The NMR spectrum data, IR spectrumdata, and UV spectrum data are shown below.

¹H-NMR (DMSO): δ=1.10–1.80 (m, 24H), δ=1.70 (s, 12H), δ=2.05 (m, 4H),δ=2.10–2.30 (m, 8H), δ=2.15 (s, 6H), δ=2.72 (s, 3H), δ=2.85 (s, 3H),δ=2.85–3.50 (m, 12H), δ=3.55 (m, 4H), δ=4.12 (t, 4H), δ=6.54 (d, 2H),δ=7.36–7.95 (m, 6H), δ=8.36 (t, 1H) IR: 2127 cm⁻¹ (—N═C═N— group) UV(EtOH): λ_(max) 532 nm

Example 4

(1) Production of Fluorescent Group-Containing Compound Represented bythe General Formula (VIII)

Cy5.18.OSu (200 mg, 0.171 mmol) produced by referring to Japanese PatentApplication Laid-open No. 9-325147 and Ratnakaer B. Mujumdar et. al.,Bioconjugate Chem., 4, 2, 105–111 (1993) was dissolved in DMF (5 ml).After the reaction solution was cooled to 0° C.,N-3-(dimethylaminopropyl)amine (70 mg, 0.684 mmol) was gradually addedthereto, and the mixture was stirred at room temperature for 12 hours.After this reaction solution was concentrated, ethyl acetate/methanolwas added thereto, and the mixture was decanted to obtain Compound(IV-I-A) (184 mg, yield: 94%). The NMR spectrum data are shown below.

¹H-NMR (DMSO): δ=1.32–1.65 (m, 12H), δ=1.72 (S, 12H), δ=2.05 (t, 4H),δ=2.00–2.30 (m, 8H), δ=2.15 (s, 12H), δ=2.95 (q, 4H), δ=4.17 (t, 4H),δ=6.57 (d, 3H), δ=7.36–7.95 (m, 6H), δ=8.45 (t, 2H)

(2) Production of Fluorescent Group-Containing Carbodiimide CompoundRepresented by the General Formula (III)

Compound (I-I-D) (82 mg, 0.157 mmol) obtained in the Example 1 (1) andCompound (IV-I-A) (180 mg, 0.157 mmol) obtained in the Example 4 (1)were dissolved in DMF (10 ml), and the solution was stirred overnight atroom temperature. To the reaction mixture, ethyl acetate/methanol wasfurther added and the mixture was decanted to obtain Compound (4) (246mg, yield: 94%) as the fluorescent group-containing carbodiimidecompound of the present invention. The NMR spectrum data, IR spectrumdata, and UV spectrum data are shown below.

¹H-NMR (DMSO): δ=1.22–1.65 (m, 12H), δ=1.72 (s, 12H), δ=2.05 (t, 4H),δ=1.95–2.30 (m, 8H), δ=2.15 (s, 12H), δ=2.95 (q, 4H), δ=4.17 (t, 4H),δ=6.57 (d, 3H), δ=7.32–7.95 (m, 6H), δ=8.45 (t, 2H) IR: 2127 cm⁻¹(—N═C═N— group) UV (EtOH): λ_(max) 632 nm

Example 5

<Water solubility Test>

Compounds (1)–(4) obtained in Examples 1–4 were tested for theirsolubility in water. For comparison, the test for solubility in waterwas also performed for Compound (1′) corresponding to Compound (1) wherethe potassium carboxylate group (—COOK) in the Rhodamine B group wasreplaced with a hydrogen atom, Compound (2′) corresponding to Compound(2) where the sodium sulfonate group (—SO₃Na) in the IRD-41 group wasreplaced with a methyl group, Compound (3′) corresponding to Compound(3) where the potassium sulfonate group (—SO₃K) in the Cy3 group wasreplaced with a hydrogen atom, and Compound (4′) corresponding toCompound (4) where the potassium sulfonate group (—SO₃K) in the Cy5group was replaced with a hydrogen atom. The results are shown in Table1.

TABLE 1 Solubility in water Compound (1) 30 μg/μl Compound (1′)  5 μg/μlCompound (2) 55 μg/μl Compound (2′) 10 μg/μl Compound (3) 95 μg/μlCompound (3′) 25 μg/μl Compound (4) 90 μg/μl Compound (4′) 10 μg/μl

From these results, it is clear that the fluorescent group-containingcarbodiimide compounds of the present invention are excellent in watersolubility.

Example 6

Reaction solutions containing 0.1 M of each of Compounds (1), (3) and(4) obtained in the Examples 1, 3 and 4, and 1 μg of phage DNA (M13 mp18replicate type, produced by Takara Shuzo Co., Ltd.) in borate buffer (pH8.5) were prepared and incubated at 85° C. for 1 minute. To eachreaction mixture, 1/9-fold volume of 3 M sodium acetate and 2.5-foldvolume of cold ethanol were added and mixed, and the mixture was left at−80° C. for 45 minutes. The mixture was centrifuged at 4° C. and 12,000rpm for 1.5 minutes by using a centrifugal machine (H-1500FR, producedby Kokusan Co., Ltd.). After the supernatant was removed, the obtainedprecipitates were dissolved in 100 μl of sterilized water.

Then, 480 ng to 480 pg/100 μl of 10-fold serial dilutions of the phageDNA (M13mp18 replicate type) linearized with a restriction enzyme(HindII) were prepared in 2 M NaCl, heat-treated for 10 minutes at 100°C., and quenched on ice for 5 minutes to obtain heat-denatured nucleicacid. The heat-denatured nucleic acids at various concentrations wereadded to wells of a plate, and immobilized at 37° C. for 12 hours withplate sealing.

After the plate with the immobilized heat-denatured nucleic acids waswashed with distilled water, 100 μl of a prehybridization solution[5×SSC (1×SSC=0.15 M NaCl, 0.015 M sodium citrate), 5× Denhardt'ssolution (0.02% polyvinylpyrrolidone, 0.2% Ficoll, 0.02% BSA), 25 mMsodium phosphate buffer (pH 6.5), 50% formamide, 0.5 mg/ml yeast tRNAand 0.1% sodium dodecylsulfate] were added to each well, and the platewas shaken by a plate mixer at room temperature for 5 minutes. Thisprocedure was repeated 3 times. The solution in each well was discarded,and 300 μl of 2×SSC was added to each well and left at room temperaturefor 5 minutes.

Then, 100 μl of 50 mM sodium phosphate buffer (pH 7.0) containing 1.5 MNaCl was added to each well to form fluorescent substance-containing DNAas Compounds (1A), (3A) and (4A). The solutions of the wells were eachtaken into a capillary tube and irradiated with an excitation light, andfluorescence emitted from the fluorescent group used to label the M13replicate type DNA was measured by a spectrophotofluorometer (F-3010:produced by Hitachi Co., Ltd.). The results are shown in Table 2.

TABLE 2 Detection Excitation Fluorescence limit wavelength wavelength(ng/well) (nm) (nm) Compound (1A) 4.3 563 583 Compound (3A) 3.2 554 574Compound (4A) 3.3 654 674

From these results, it can be seen that detection of nucleic acid can beperformed with high sensitivity according to the method for detection ofnucleic acid of the present invention.

1. A fluorescent group-containing carbodiimide compound precursor havinga halogen atom or a sulfonic acid group which is represented by thefollowing general formula (I):B—Y³—N═C═N—Y²—W—Y¹-{A}_(n)-Y⁴-x  (I) Wherein, X represents a halogenatom or a sulfonic acid group; A represents a functional group selectedfrom the group consisting of —CH₂—, —NHCO—, —CONH—, —O—, —S—, —NR¹—wherein R¹ represents a linear, cyclic or branched saturated orunsaturated aliphatic hydrocarbon group having 1–20 carbon atoms,—NR²R³— wherein R² and R³ each independently represent a hydrogen atom,a linear or branched saturated or unsaturated aliphatic hydrocarbongroup having 1–20 carbon atoms, or a cycloalkyl group, an aryl group oran aralkyl group which may have a substituent, provided that when one ofR² and R³ is a hydrogen atom, the other represents a linear or branchedsaturated or unsaturated aliphatic hydrocarbon group having 1–20 carbonatoms, or a cycloalkyl group, an aryl group or an aralkyl group whichmay have a substituent, or R² and R³ may be bonded to each other to formas a whole a nitrogen-containing heterocyclic group which may contain anoxygen atom, —COO—, —OCO—, —NHSO₂—, —NHC(S)NH— and SO₂NH—; n represents0 or 1; W represents a direct bond or a quaternary onium group; Y¹, Y²,Y³ and Y⁴ each represent a functional group represented by the generalformula (II):—(CH₂)_(p)-(L)_(r)-(CH₂)_(q)—  (II) wherein, L represents a functionalgroup selected from the group consisting of —CH₂—, —NHCO—, —CONH—, —O—,—S—, —NR¹— wherein R¹ has the same meaning as defined for the formula(I), —NR²R³— wherein R² and R³ have the same meanings as defined for theformula (I), —COO, —OCO—, —NHSO₂—, —NHC(S)NH— and SO₂NH—; p and q eachrepresent an integer of from 0 to 20; and r represents 0 or 1; Brepresents a hydrogen atom or a monovalent organic group; and Any of thefunctional groups represented by B, Y¹, Y², Y³, Y⁴, A and W may containa group selected from a carboxyl group, a sulfo group, a phosphono groupand a phospho group which have substitution of an alkali metal, analkaline earth metal or a basic group containing a nitrogen orphosphorus atom.
 2. The fluorescent group-containing carbodiimidecompound precursor according to claim 1, wherein at least one functionalgroup selected from B, Y¹, Y², Y³, Y⁴, A and W in the formula (I) has atleast one group selected from a carboxyl group, a sulfo group, aphosphono group and a phospho group which have substitution of an alkalimetal, an alkaline earth metal or a basic group containing a nitrogen orphosphorus atom.